Chirality, also known as handedness or left-right (LR) asymmetry, is a conserved feature in the development of multi-cellular organisms, and can be seen in the growth of climbing plants, the helices of snail shells, and the positioning of internal organs in the human body. For human, defects in laterality such as isomerism (loss of asymmetry), and heterotaxia (a loss of concordance among the individual organs) are observed in more than 1 in 8000 live births, and have significant clinical implications. In vertebrates, epithelial chiral morphogenesis is important in establishing the LR asymmetric body plan, from the early nodal flow at the ventral node to the later heart c-looping and gut asymmetric rotation. Traditionally, LR asymmetry is studied with animal embryos in vivo, which is often very challenging. The direct manipulation of human embryos is restricted because of the obvious ethical concerns. Recently, we have recapitulated epithelial chiral morphogenesis on micropatterned surfaces. Now we want to further develop in vitro systems for studying embryonic LR axis development. Our rationale is that novel cell chirality based high-throughput platforms and a better understanding of molecular mechanisms of epithelial cell chirality can greatly facilitate the LR asymmetry research in developmental biology. We propose to use a combination of embryonic stem cell culture, micro-fabrication, live cell imaging, molecular assay, traction force measurement, and high-throughput screening as tools to elucidate the underlying biophysical and biochemical mechanisms for epithelial chiral morphogenesis. Our objectives are to establish multiscale in vitro models for LR asymmetry in development and to identify important signaling pathways and cytoskeletal proteins that affect epithelial cell chirality. Specific Aim 1 (SA1): Establish and optimize multiscale in vitro models for studying LR asymmetry in development. We propose to improve our 2D multicellular model by matching substrate stiffness and ligand type with that of native embryonic tissue and to extend to models at single cell level and 3D tubular cell sheet. Specific Aim 2 (SA2): Determine effects and mechanisms of stem cell differentiation, growth factors, drugs that are important for LR asymmetry. We aim to establish a link between patterned epithelial cell chirality and developmental LR asymmetry. Specific Aim 3 (SA3): Determine cellular machinery mechanisms in patterned cell chirality. This will allow us to identify the ultimate cellular machinery for the emergence of chiral morphogenesis. Overall, if we are successful, these studies will establish novel, paradigm-shifting systems for measuring cell chirality in a high throughput fashion for studying LR asymmetry in development and disease, and screening genetic and biochemical factors that cause birth defects. In addition, this proposed research is transformative, and potentially open a new field of research: cell chirality, a fundamental cellular property defining the third axis of the cell.